Many animal cells used for the production of viral vaccines, growth factors, receptors or therapeutic proteins are anchorage-dependent, i.e. they must adhere to a compatible surface in order to grow. This requirement is particularly stringent for normal diploid cells; these cells not only need to attach to a surface, they develop a polarized, elongated cell shape after attachment and eventually grow to cover the surface and reach a state of confluence. At confluence cell division stops. Contact-inhibition stays further cell growth. These cells form a monolayer of cells on a surface. Cell growth resumes only after the cells are detached by exposure to a proteolytic agent, such as trypsin, and plated onto a larger surface.
Transformed cells and continuous (non-diploid) cell lines, on the other hand, often retain the requirement of surface for growth but lose the characteristic of contact inhibition. Such cells may form multiple layers of cells on a surface if proper growth medium is available. (See Hu, W-S. and Dodge, T. C. (1985), "Cultivation of Mammalian Cells In Bioreactors", Biotechnol. Prog. 1, 209-215). Although some transformed cells acquire the ability to grow in suspension, many of these cells preferentially adhere to a surface if a compatible surface is available.
Conventionally, cells have been cultivated in roller bottles. Since the 1980's, the demand for large quantities of therapeutic proteins, such as tissue type plasminogen activator, has resulted in a wider application of many alternative cultivation methods which are more suitable for large-scale operations. These alternative methods include microcarriers based on dextran (See Van Wezel, A. L. (1967), "Growth of Cell Strains and Primary Cells on Microcarriers in Homogenous Culture", Nature 216:64:65); polystyrene (See Johansson, A. and Nielsen, V. (1980), "Biosilon: A New Microcarrier", Dev. Biol. Stand 46:125-129 and Kuo, M. J., Lewis, C. Jr., Martin, R. A., Miller, R. E., Schoenfeld, R. A., Scheck, J. M. and Wildi, B. S. (1981), "Growth of Anchorage-Dependent Mammalian Cells on Glycine-Derivatized Polystyrene in Suspension Culture", In Vitro 17:901-906); cellulose (See Reuveny, S., Silberstein, L., Shahar, A., Freeman, E. and Mizrahi, A. (1982), "Cell and Virus Propagation on Cylindrical Cellulose Based Microcarriers", Dev. Biol. Stand. 50:115-123); collagen (See R. C. Dean et al. (1985), Large Scale Mammalian Cell Culture Technology. Ed. B. K. Lydersen, Hansen Publishers, New York, N.Y., pp. 145-167); gelatin-based macroporous beads (See Cultisphere, Technical Bulletin, Percell Biolytica AB); hollow-fiber bioreactors (See Knazek, R. A., Gullino, P. M., Kohler, P. O. and Dedrick, R. L. (1972), Science 178:65); and ceramic bioreactors (See Lydersen, B. K., Pugh, C. G., Paris, M. S., Sharma, B. P. and Noll, L. A. (1985), Biotechnology 3:63).
Among these techniques, microcarrier technology is the most widely employed, especially in vaccine production. The mean diameter of microcarriers reported in the literature are generally in the range of 130 .mu.m to 200 .mu.m. (See, e.g., Hu, W-S., Meier, J. and Wang, D. I. C. (1985), "A Mechanistic Analysis of The Inoculum Requirement For The Cultivation of Mammalian Cells on Microcarriers"; Biotechnol. Bioeng. 27, 585-595; Varani, J., Dame, M., Fediske, J., Beals, T. F. and Hillegas, W. (1985), "Substrate-Dependent Differences in Growth and Biological Properties of Fibroblasts And Epithelial Cells Grown in Microcarrier Culture", J. Biol. Stand. 13:67-76; Microcarrier Culture, Pharmacia Fine Chemicals Technical Bulletin; Blasey, H. D., Grossebueter, W., Lehman, J., Giehring, H. and Schwengers, D. (1988), "B-interferon Production in Serum-Free Medium On A New Type Of Microcarrier", Presentation at the Engineering Foundation on Cell Culture Engineering, Palm Coast, FL, U.S.A., Jan. 31-Feb. 5, 1988.). However, a range as wide as from 100 to 400 .mu.m has been said to be suitable for growth. (See, e.g., Butler, M. (1987), "Growth Limitation In Microcarrier Culture", Adv. Biochemical Engineering/Biotechnology 34:57-84.). It should be noted that the diameter of many types of microcarriers change with the tonic strength and pH of the solution in which they are suspended. In this patent application, the diameter measured in a phosphate buffer saline or cell culture media will be referred to as the hydrated diameter.
Because most types of microcarriers have an apparent density slightly heavier than water (1.02-1 g/cm.sup.3), a conventional stirred-tank, or fermentor, is used to keep microcarriers in suspension. Once suspended, cells attach, spread and grow on the external surface of the microcarriers. The concentration of microcarriers used generally ranges from an equivalent of 2% of the settled bead volume to 12% of the settled bead volume. Although as high as 40% of settled bead volume has been used, it is not common practice. (See Hu, W-S., Meier, J. and Wang, D. I. C. (1985), "A Mechanistic Analysis of The Inoculum Requirement For The Cultivation of Mammalian Cells on Microcarriers", Biotechnol. Bioeng. 27, 585-595.).
A major advantage of microcarrier technology is the large amount of available cell growth area (0.4 m.sup.2 /l to 3 m.sup.2 /l) that can be contained in the reactor vessel. A drawback of microcarrier technology, on the other hand, is the large amount of settled bead volume per unit volume of the reactor. Virtually all this volume is inert; except for providing growth surface, the settled bead volume is not contributing to cell growth and product formation. The relatively large amount of settled bead volume also increases the degree of sophistication required for the design of an agitation system to suspend the beads without damaging the cells. (See Hu, W-S. and Wang, D. I. C. (1986) "Mammalian Cell Culture Technology: A Review From An Engineering Perspective", Mammalian Cell Technology. Ed. W. G. Thilly, Butterworths Publishing Company.).
In principle, it might appear that the diameter of microcarriers could be decreased to increase the cell growth surface area per unit volume of microcarriers. The external surface per unit volume of microcarriers increases linearly with decreasing diameter. Furthermore, it has been proposed that smaller beads have a lower frequency of collision and a lower kinetic energy, and may reduce possible collision damage of cells in a bioreactor. (See Cherry, R. S. and Papoutsakis, E. T. (1986) "Hydrodynamic Effects On Cells In Agitated Tissue Culture Reactor" (Bioprocess Eng. 1:29-41)).
Despite the possible benefits of smaller microcarrier beads published reports state that the minimum optimal diameter range for the selected microcarrier is between 90-100 .mu.m. It has ben speculated that it is a disadvantage to have cells adhere to a more curved surface such as the surface available on smaller diameter beads. (Id.). This belief is consistent with a previous report on cell growth on glass fiber which appears to indicate that curvature of surface plays a role in how cells grow.
As a result of these findings, the diameter range of commercially available microcarriers fall in the range of the reported "optimal" diameter range of 90-200 .mu.m. (See Hu, W-S. and Wang, D. I. C. (1987), "Selection of Microcarrier Diameter For The Cultivation of Mammalian Cells on Microcarriers", Biotechnol. Bioeng. 30, 548-555; Kubota, H. and Nagaike, K. (1989), "Cell Culture Using Microcarriers: The Effect of Chemical And Physical Properties of Microcarrier on Cell Attachment, Spreading and Growth", Abstract and presentation at the Japanese Association of Animal Cell Technology annual meeting, Tsukuba City, Japan, Nov. 20-22, 1989.).
Similarly, chick fibroblasts cultivated on glass wool fibers have been observed to spread along different directions on the fiber if the fiber diameter is larger than 100 .mu.m. When cultivated on fibers of about 70 .mu.m, they spread and grow only along the longitudinal direction. (See Fisher, P. E. and Tickle, C. (1981), "Differences in Alignment Of Normal and Transformed Cells on Glass Fibers", Exp. Cell Res. 131:407:410.).
Furthermore, cells attached to small beads do not develop their typical morphology as they do on a flat surface. Exceptions to the aforementioned optimum diameter range do exist. For example, when celluloid materials were used, such as DE-52 and DE-53, the diameter of rods was approximately 40 .mu.m or longer, up to hundreds of .mu.m. (See Reuveny, supra). In another case, small polystyrene beads BioBeads SX-1 of 25 .mu.m were used. These beads failed to support significant cell growth (only 60% increase in DNA content over a 14-day period). However, other polystyrene beads of unspecified diameter and only after some surface modifications, did support cell growth.
To circumvent the drawbacks of microcarriers, some have opted to use cells which can be adapted to grow in suspension. Chinese hamster ovary (CHO) cells can be used in this fashion. (See Murata, M., Eto, Y. and Shibai, H. (1988), "Large-Scale Production of Erythroid Differentiation Factory By Gene-Engineered Chinese Hamster Ovary (CHO) Cells in Suspension Culture", J. Ferment. Technol. 66:501-507.). However, the use of suspension cells suffers another shortcoming: the maximum cell concentration achievable in a conventional stirred-tank bioreactor is only in the vicinity of 2.times.10.sup.6 /ml. To achieve a high cell concentration, the medium has to be replenished intermittently or perfused continuously to supply nutrients and to remove metabolites. In a microcarrier culture, on the other hand, cells on microcarriers can be retained in the bioreactor by sedimentation or by withdrawing the medium through a rotating sieve. In a suspension culture, cell retention in the bioreactor poses some difficulty, and continuous or intermittent medium replenishment inevitably removes cells along with the spent medium.
An approach recently adopted to overcome this problem is to cultivate some transformed cells as cell aggregates. (See Tolbert, W., Hitt, and Feder, J. (1980), "Cell Aggregate Suspension Culture", In Vitro 16:486:490.). Different methods have been used to induce the aggregate formation for cells which normally grow on surface or prefer to grow on surface. This method usually involves the use of medium containing a low concentration of calcium in conjunction with a moderately high agitation rate. The aggregate method of cell cultivation allows for easier cell retention due to the larger size of the particles. Thus, a high cell concentration can potentially be achieved. Compared with a microcarrier culture, the settled volume of the solid phase (cell mass or beads) is much lower than the same cell concentration in an aggregate culture.
The sizes of cell aggregates span widely from single cells to very large aggregates of hundreds of .mu.m in diameter. It is believed that throughout the cultivation period single cells and small aggregates continuously adhere to one another to form new or larger aggregates, while larger aggregates continuously break down to generate smaller ones. With such a wide range of aggregate size, the cultivation conditions using simple suspension culture techniques are hardly optimum. Large aggregates, as large as 1 mm, may suffer nutrient transfer limitations in their interiors, while small aggregates may easily be washed out in a continuous flow reactor. Therefore, it is not surprising that despite its high total cell concentration the increase in volumetric productivity of an aggregate culture is often not proportional to the increase in cell concentration from that of a conventional continuous flow reactor.
Furthermore, only cells which are capable of growing in suspension, such as CHO cells (See Karkare, S. B., Webster, J. and Tajiri, D. (1988), "Modeling of Continuous Culture of Clumped CHO Cells For the Production of Erythroprotein", Abstract and Presentation at the Engineering Foundation on Cell Culture Engineering, Palm Coast, FL, U.S.A., January 31-February 5, 1988); or highly transformed cells such as CRL 598 cells (See Avgerinos, G. C. and Drapeau, D. (1988), "Production Scale Mammalian Cell Suspension Perfusion Culture With a Rotating Wire Mesh Screen", Abstract and presentation at the Engineering Foundation on Cell Culture Engineering, Palm Coast, Fla., U.S.A., Jan. 31-Feb. 5, 1988); can be grown in aggregate form in a suspension culture.
Conventional microcarriers have also been used to generate cell clumps or aggregates. In the study described by G. C. Avgerinos, D. Drapeau, J. S. Socolow, J. I. Mao, K. Hsiao and R. J. Broeze, "Spin Filter Perfusion System For High Density Cell Culture: Production Of Recombinant Urinary Type Plasminogen Activator In CHO Cells", Bio/Technology 8:54-58, Chinese hamster ovary cells were cultivated in conventional microcarriers (Cytodex 2, Pharmacia) and grow normally. After the cells reached confluence, the cells began to migrate toward one end of the microcarriers and began to form clumps. After 18 days, some large aggregates with diameters in the range of 200 to 600 .mu.m were observed.
Similarly, in other studies, it has been reported that certain cells cultivated on cellulose rods formed aggregates which contained a number of cellulose rods and cells bridged between these rods. (See J. Litwin, "The Growth Of Human Diploid Fibroblasts As Aggregates With Cellulose Fibers In Suspension", Dev. Biol. Standard. 60:237-242 (1985) and Kotler, M. Reuveny, S., Misrahi, A. and Shahaz, A, "Ion Exchange Capacity Of DEAE-Microcarriers Determined The Growth Pattern Of Cells In Culture", Dev. Biol. Standard. 60:255-261 (1985).). In these studies, using conventional microcarriers (Cytodex 2 and cellulose), a large amount of microcarrier was used.